The Combined Effect of Dissolved Organic Carbon and Salinity on the

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The Combined Effect of Dissolved Organic Carbon and Salinity on the Bioaccumulation of Copper in Marine Mussel Larvae David Deruytter,*,† Jan Garrevoet,‡ Michiel B. Vandegehuchte,† Eva Vergucht,‡ Björn De Samber,‡ Bart Vekemans,‡ Karen Appel,§ Gerald Falkenberg,§ Katrien Delbeke,∥ Ronny Blust,⊥ Karel A. C. De Schamphelaere,† Laszlo Vincze,‡ and Colin R. Janssen† †

Laboratory of Environmental Toxicology and Aquatic Ecology, Department of Applied Ecology and Environmental Biology, Ghent University, Jozef Plateaustraat 22, B-9000 Gent, Belgium ‡ X-ray Microspectroscopy and Imaging Group, Department of Analytical Chemistry, Ghent University, Krijgslaan 281, B-9000 Gent, Belgium § Hamburger Synchrotronstrahlungslabor (HASYLAB) at the Deutsches Elektronen Synchrotron (DESY), Notkestrasse 85, D-22607 Hamburg, Germany ∥ European Copper Institute, Avenue de Tervueren 168 b10, B-1150 Brussels, Belgium ⊥ Systemic Physiological & Ecotoxicological Research, Department of Biology, University of Antwerp, Groenenborgerlaan 171/U7, B-2020 Antwerp, Belgium S Supporting Information *

ABSTRACT: Larvae of Mytilus spp. are among the most Cu sensitive marine species. In this study we assessed the combined effect of salinity and dissolved organic carbon (DOC) on Cu accumulation on mussel larvae. Larvae were exposed for 48 h to three Cu concentrations in each of nine salinity/DOC treatments. Synchrotron radiation X-ray fluorescence was used to determine the Cu concentration in 36 individual larvae with a spatial resolution of 10 × 10 μm. Cu body burden concentrations varied between 1.1 and 27.6 μg/g DW larvae across all treatments and Cu was homogeneously distributed at this spatial resolution level. Our results indicate decreasing Cu accumulation with increasing DOC concentrations which can be explained by an increase in Cu complexation. In contrast, salinity had a nonlinear effect on Cu. This cannot be explained by copper speciation or competition processes and suggests a salinity-induced alteration in physiology.



INTRODUCTION Copper concentrations can be elevated in the aquatic environment both due to natural and anthropogenic influences and this can result in adverse effects on aquatic organisms. However, the total dissolved Cu concentration is, by itself, an inefficient predictor of those effects.1 Considerable efforts have been made to improve the prediction of toxic effects by taking into account the aquatic concentration of other elements and dissolved organic carbon (DOC). One widely used model to predict the accumulation and toxicity of metals including Cu is the biotic ligand model (BLM).2 A first BLM principle states that the bioavailability of Cu, which determines Cu accumulation at the biotic ligand, depends on the water chemistry.1 This can occur through complexation with organic or inorganic substances (e.g., DOC or inorganic anions) or by competition with cations (H, Na, Ca2,...) for binding with the biotic ligand.1 When Cu2+ activity instead of the total dissolved Cu concentration is used to © 2013 American Chemical Society

predict Cu accumulation, factors modifying complexation (anions and DOC) should not influence Cu accumulation predictions any more. In that case, only factors modifying competition at the biotic ligand (other cations) need to be accounted for. According to a second BLM principle, the Cu concentration at the biotic ligand is directly related to the magnitude of the adverse effects of Cu.1 This implies the existence of a critical Cu biotic ligand concentration, independent of the water chemistry or the organisms’ physiology. In the first BLMs developed for freshwater fish, the gills represented the biotic ligand.2 However, experimentally evaluating the accumulation of metals at the biotic ligand of much smaller animals can be problematic as it is difficult to Received: Revised: Accepted: Published: 698

June 3, 2013 November 22, 2013 December 5, 2013 December 5, 2013 dx.doi.org/10.1021/es4024699 | Environ. Sci. Technol. 2014, 48, 698−705

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Measuring the copper body burden and distribution in mussel larvae is challenging due to their small size (approximate shell length 106 μm20). However, this information is necessary to develop a BLM. In this study, we used synchrotron radiation X-ray fluorescence (SR-XRF) microspectroscopy to quantify the copper concentration and its spatial distribution in individual larvae. SR-XRF microanalysis has been successfully used to determine the concentration and distribution of metals in various biological tissues such as plants (Iberis intermedia), animals (Daphnia magna), and humans (breast tumors).21−24 Although this technique can analyze the concentration and distribution simultaneously at low concentrations (ng/g), at a high resolution (a few hundred nm)25,26 and in a nondestructive manner, the use of SR-XRF microanalysis in ecotoxicology is scarce.27,28 The primary goal of the present study was to assess concurrently the influence of DOC and salinity on the accumulation and distribution of copper in mussel larvae (Mytilus galloprovincialis). With this knowledge we evaluated whether the observed accumulation results were in agreement with the BLM principles and if whole larvae can be used as biotic ligand. The secondary goal was evaluating the potential use of microbeam and nanobeam SR-XRF measurements in a full-scale experiment with small multicellular organisms for assessing metal accumulation.

determine concentrations in different tissues and the actual site of toxic action is often not known. Furthermore, previous research has demonstrated that the relationship between the total body residue of metals and their adverse effects is not always clear.3 This may be due to the fact that not all accumulated metal is metabolically available and consequently does not contribute to toxicity.4 In a freshwater environment, alterations in the concentration of DOC, Na, Ca, etc. can all alter the toxicity of Cu.1,5 The adverse effects of Cu exposure to fish in freshwater media with different chemical compositions can be accurately predicted by taking speciation and competition into account, thus indicating that a BLM approach can be used in a freshwater environment.6 In marine and estuarine environments, research to elucidate the influence of ions and DOC on metal bioavailability is scarce. From the available literature it seems that at least DOC can significantly reduce the accumulation and toxicity of Cu in a similar manner as that observed in freshwater systems.7−9 In contrast to freshwater, in natural seawater the pH is buffered and the ratios between the different ion concentrations are relatively constant. Hence, pH and the effect of changes in the concentration of single ions are most probably less important for a marine and estuarine BLM and are therefore not considered in this study. Although the ion concentration ratios in seawater are constant, the absolute ion concentrations change with changing salinities. According to the BLM concept, a higher salinity (ion concentration) should reduce metal accumulation at the biotic ligand due to the effect of ionic strength on the activity of the free metal ion and others species, increased complexation with inorganic anions (e.g., CuCl2 , CuCO3 ) and increased competition with other cations (e.g., Na) at the biotic ligand. In some cases a change in Cu2+ activity with changing salinities can explain the salinity-dependent differences in toxicity.9,10 However, the effect of salinity on metal toxicity and accumulation remains unclear. Previous research indicated that the relationship between Cu accumulation and salinity may not be similar for all organisms and may depend on several factors.10−12 There is substantial evidence that, besides bioavailability, alterations in the organism’s physiology caused by salinity differences are important to explain changes in Cu accumulation and/or toxicity.10,11,13−15 Currently the BLM does not account for changes in physiology.16 Mytilus spp., especially their larvae, are very sensitive to Cu and are both ecologically and economically important. This makes them ideal species for the further development of a marine/estuarine copper BLM. By using a freshwater BLM (version AP08, build 2001−10−02) the protective effect of DOC on the toxicity of Cu in mussel larvae has successfully been predicted by Arnold et al.17 Rosen et al.9 found a clear relationship between the external Cu concentration and Cu accumulation in M. galloprovincialis larvae, demonstrating a decrease in accumulation and toxicity with increasing DOC, confirming the findings of Arnold et al.17,18 Rosen et al.9 found a critical body Cu residue of 49 μg/g dry weight (DW) larvae at which 50% of the larvae was deformed after 48 h, independent of the DOC concentration. However, differences in salinity between tested media were small or absent in both studies. As such, to date the effect of a broad range of salinities (e.g., 4−38 psu in the Adriatic sea19) on the accumulation of Cu in mussel larvae is still unknown. Furthermore, it is unknown whether salinity affects the influence of DOC on Cu accumulation.



MATERIALS AND METHODS Adult Mussel Collection and Maintenance. Adult Mytilus galloprovincialis (n = 150, approximately 5 cm) were collected in the bay of Venice (Italy, March 2012). The ambient salinity was 35 psu and seawater temperature was 8 °C. The mussels were transported overnight by plane in an insulated box. The box was filled with ice to keep the mussels cool and avoid spontaneous spawning due to a temperature rise. Upon arrival in the laboratory the mussels were randomly divided in three groups, cleaned and placed in recirculating, aerated, artificial seawater (Instant Ocean) at a temperature between 8 and 10 °C, a salinity of 35 psu and fed ad libitum with Shellfish Diet 1800 (Reed Mariculture Inc.). Salinity was altered in the first week after arrival at a rate of 1 to 2 psu/day until the desired salinity for the spawning experiment was reached (24, 30, or 36 psu; Supporting Information (SI) Figure S2). The mussels were left to acclimate to the final conditions for another week. Experimental Design. The experiment was designed to simultaneously assess the effect of salinity and DOC concentration on copper accumulation in mussel larvae. Mussel embryos were exposed for 48 h to nine combinations of salinity and DOC, based on a central composite design. In that design there is a middle point, four corner points and four star points as extremes (Table 1, SI Figure S1). The measured salinity ranged between 22.9 and 36.8 psu and the DOC concentration between 0.56 and 4.66 mg/L. Exposure media were prepared by combining natural estuarine water, artificial seawater29 and deionized water to achieve the required salinity/DOC combination. Natural estuarine water (filtered 0.2 μm) was collected in Nieuwpoort (Belgium) and used as a source of natural DOC. A preliminarily test demonstrated that this water did not cause adverse effects on the larval development (>95% normal development). In every salinity/DOC treatment, embryos were exposed to a control without added Cu and five different Cu concentrations. The Cu concentrations were chosen based on literature data on Cu toxicity in mussel 699

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from adults acclimated to 24 psu were used for the three lowest salinities (22.9, 24.7, and 25.1 psu). Similarly, embryos from adults acclimated to 36 psu were used the highest salinities (34.7, 34.9, 36.8 psu, SI Figure S2). Analytical Chemistry. Water samples for chemical analyses were taken at the start the experiment. Samples were also collected at the end for part of the Cu concentrations (control, middle, and highest exposure concentration) to determine the change in Cu and DOC over time. Samples from the four replicates were pooled and filtered (0.45 μm). The samples for metal analysis were acidified with 0.14 mol/L analytical grade HNO3 and stored in polypropylene tubes at 4 °C before analysis. DOC samples were stored in glass tubes at 4 °C ensuring no air was left in the tube. DOC analysis was performed with a Shimadzu TOC-5000 analyzer using the hightemperature catalytic oxidation (HTCO) technique.33,34 The statistical analysis was performed on the mean DOC concentration of each salinity/DOC combination. At the end of the test, salinity was measured with a WTW cond 315i (tetracon 325 electrode) and the pH was measured with a Knick portamess (InPro 4260 electrode). Measurement of dissolved Cu was performed using ICP-MS. A high resolution ICP-MS instrument (Thermo Element 2 XR) was used to determine the metals after appropriate dilution and acidification of the water to reduce salt levels. Instrument settings were used that provided optimal resolution and sensitivity for the given matrix and elements. The Cu concentration at the start of the 48 h exposure period was used for further statistical analysis as recommended by the ASTM.29 After 48 h on average 32% of the Cu was present at a mean initial concentration of 1.00 μg Cu/L (control), 66% at a mean initial concentration of 6.85 and 92% at a mean initial concentration of 21.4. The activity of Cu2+ and other Cu species were calculated using Visual MINTEQ 3.035 with the specific interaction theory (SIT) activity correction. Input values were based on measured salinity, pH, DOC and

Table 1. Different Combinations of Salinity, DOC, Cu and the Group (Day) They Were Tested and Their Position in the Central Composite Design; S = Star Point, C = Corner Point, M = Middle Point group

place

salinity (psu)

DOC (mg/L)

Cu 1 (μg/L)

Cu 2 (μg/L)

Cu 3 (μg/L)

1 2 2 1 1 1 2 2 2 1

S C C M S S M C C S

22.9 24.7 25.1 29.45 29.8 29.8 29.95 34.7 34.9 36.8

3.00 1.24 3.92 3.02 0.56 4.66 2.80 4.56 1.46 2.98

0.91 0.78 1.79 1.025 0.03 1.71 Lost 1.28 0.80 1.01

3.87 3.66 4.30 4.22 2.16 4.45 3.44 3.46 3.07 7.69

12.44 8.98 12.21 11.6 5.25 12.59 11.46 10.97 5.39 10.94

larvae18,30 and are environmentally realistic.31 Two Cu concentrations were selected out of the five tested concentrations based on visual observation of the larval development. To ensure equilibrium between Cu and seawater, poly ethylene vials (50 mL) were filled with 40 mL of the appropriate medium and spiked with copper (as dissolved CuCl2) one day prior to use.32 Each treatment was replicated four times. Due to logistic restrictions the experiment was split in two groups consisting of 5 salinity/DOC combinations and set up on two consecutive days (Table 1, SI Figure S1). The middle point (±30 psu, 2.5 mg DOC/L) was replicated each day to account for possible day to day variation. 48 h Embryo Exposures. Spawning was performed according to the ASTM protocol E724−9829 with the following adjustment: to determine the influence of salinity on the copper accumulation, spawning of the mussels and exposure or the embryos was performed in seawater with the desired salinity (SI Figure S1) and not at the proposed salinity of 34 psu. Embryos

Figure 1. Examples of the μSR-XRF data at a resolution of 10 × 10 μm (scale bare bottom right). A: optical image of the larva; B−D: Cu concentration image of mussel larvae exposed to a salinity of 29.8 psu, a low DOC concentration (0.56 mg/L) and different dissolved Cu concentrations (increasing from left to right); E: result of the clustering analysis with larva, glue, carbon tip and mixture regions; F−H: Cu concentration of mussel larvae exposed to the same salinity (29.8 psu) but a higher DOC concentration (4.66 mg/L) and different dissolved Cu concentrations (increasing form left to right). Darker pixels indicate a higher Cu body burden (see legend on the right). The high Cu concentration at the bottom (dark region) of each image is the result of Cu in the carbon tip which held the larvae. 700

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and the obtained reference spectra were analyzed using the same data treatment method as that applied for the unknown samples to ensure a correct quantification. Furthermore, the SRM measurements are used for the determination of the minimum detection limits (MDL, SI Figure S3). A MDL is defined as the amount of analyte that causes a net peak intensity equal to three times the standard counting error of the background intensity. The Cu body burden is defined as the total concentration of Cu present in the soft tissues and/or shell of the larvae. No distinction could be made between these two compartments. Although some other studies used EDTA to remove loosely bound Cu on the shell, we assume that eight washing suffice steps as other studies on mussel larvae used a single washing step with deionized water.9,20 Synchrotron Radiation X-ray Fluorescence (SR-XRF) Nanoanalysis. To determine the elemental distribution in mussel larvae at a (sub) μm scale, an additional experiment was performed by exposing mussel larvae (M. edulis) for 48 h to a nominal concentration of 10 μg/L Cu in artificial seawater at 32 psu,29 without added DOC. The SR-XRF nanoanalysis was performed at the Micro/Nanoprobe Beamline P06 of the thirdgeneration synchrotron radiation facility PETRA-III (DESY, Germany). This undulator beamline is equipped with a Si111 crystal monochromator providing a spectra bandwidth of ΔE/E = 0.014% and was set at an energy of 10 keV. The primary Xray beam was focused using Fresnel Zone plates, providing a beamsize of 50 (H) by 50 (V) nm fwhm. Due to the limited available measurement time, which is typical at synchrotron sources, and the drastic increase in measurement time when measuring samples with nanometer resolution only one larva could be measured. It was not feasible to measure all 29 larvae with this technique. Statistical Analysis. Statistical analysis was performed using R 2.12.1 statistical software.40 There were no significant differences between the two parts of the experiment, therefore the random variable ‘group’ was not included in further statistical analysis. A linear accumulation model was constructed starting from the full model (M.1) with total dissolved Cu, salinity, and DOC.

measured total dissolved copper concentration. The Stockholm humic model (SHM) was used as model for the Nieuwpoort estuary DOC according to Ndungu36 with an active (Dissolved Organic Matter)/(Dissolved Organic Carbon) ((DOM)/(DOC)) ratio of 2 with 100% of the active DOM as fulvic acid. Synchrotron Radiation X-ray Fluorescence (SR-XRF) Microanalysis. One or two larvae exposed to the control or the two Cu concentrations of each salinity/DOC combination were analyzed using microbeam SR-XRF. The larvae were prepared according to the following procedure. Exposed, live 48 h old D-larvae were dehydrated using a graded acetone/water series (70%, 80%, 90%, 2 × 98%, 2 × 100% acetone) by submersing a number of larvae consecutively in the different solutions in an acid-washed (15% HCl) watch glass for 5 min. Subsequently, the larvae were submersed in hexamethyldisilazane (HMDS) for 30 min and left to dry overnight in a desiccator.37 One well developed and well preserved D-larva was and glued on the sharpened tip of a 0.5 mm carbon rod and preserved in a closed dust free container at room temperature (Figure 1A). This was done for a total of 36 larvae. One for each Cu/salinity/DOC combinations (n = 29, table 1) and a replicate for seven combinations to assess the variability. Due to time restrictions not all combinations could be tested twice. Previous experiments had indicated that it was impossible to use SR-XRF on undeveloped or heavily deformed larvae due to a poor preservation of the samples. SR-XRF microanalysis was performed during two experiments at Beamline L of the DORIS-III (HASYLAB) storage ring (4.45 GeV positron ring) at the Deutsches Elektronen Synchrotron (DESY) facility in Hamburg, Germany. During the micro-XRF experiments a Ni/C multilayer monochromator was used to obtain quasi monochromatic excitation energies of 12.5 and 12.7 keV with a spectral bandwidth of ΔE/E = 1.83%. The primary beam was focused using a polycapillary based optic, providing a beam size of 36 and 23 μm fwhm (full width at half-maximum) respectively. Parameter settings for the synchrotron micro-XRF measurements during the two beam times are listed in SI Table S1. Each larva was mounted on a goniometer head (device used to correct for sample mounting errors in XYZθφ) to position it vertically with respect to the central axis of the X-ray microbeam. To minimize self-absorption effects, the sample was placed under a 45° angle with respect to the primary beam. Spectral deconvolution (background removal, elimination of peak overlap and peak-area determination) of each individual XRF spectrum was done using AXIL (analysis of X-ray spectra by iterative least squares).38 Elemental distribution images were obtained based on the fitted net-peak intensities, subsequently analyzed by K-means clustering algorithms39 using the in-house developed software MICROXRF2. Before cluster-analysis could be performed, a square root data pretreatment was performed to take into account Poisson counting statistics with a normalization to give an equal weight to each elemental constituent included in the K-means clustering algorithm. This resulted in a correct classification of air, halo, larva, carbon tip, or a combination of previously mentioned areas (see Figure 1E). The halo, which is proportional to the concentration in the larvae and clearly visible in Figure 1D, originates from the beam profile and the 45 deg. geometry. The sum-spectra of the obtained clusters were normalized by their respective Compton scattering signal to account for differences in illuminated sample mass. For quantification purposes, a pressed pellet of NIST SRM 1577C (areal density 21.9 mg/cm2) was measured

E[Cu accumulation|Cu, salinity, DOC] = salinity + [DOC] + Cu + Cu × salinity + Cu × [DOC]salinity × [DOC] + Cu 2salinity 2 + [DOC]2

(M.1)

E[Cu accumulation|Cu,salinity,DOC] in model 1 stands for the expected accumulation of a Cu on a given set of Cu concentration, salinity and DOC concentration. This model was reduced via backward selection. Two commonly used approaches were assessed to determine the optimal model, that is, selecting the model with only significant factors remaining or the model with the lowest Akaike information criterion (AIC). The latter measures the goodness-of-fit and model complexity and can include nonsignificant terms.41 Cu body burdens and Cu2+ activity data were log10 transformed to normalize the data. A Shapiro Wilk test confirmed that both data sets were normally distributed after transformation (P-value >0.05). In the analysis based on Cu2+ activity, DOC was not included due to the strong influence of DOC on Cu2+ activity and therefore cannot be seen as an independent variable. Although we realize that salinity can influence Cu2+ activity there was no correlation 701

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between the two variables in our experiment (SI Figure S4), therefore salinity was not excluded from the model.



RESULTS K-means clustering of the μSR-XRF results successfully segregated the background, the larva, the glue, the carbon tip and larvae/air mixture pixels (Figure 1E). Additional SR-XRF measurements indicate that the Cu concentration in the glue used to hold the larvae onto the carbon rod was below the MDL (see SI; Figure S5). At a spatial resolution level of 10 × 10 μm the Cu concentrations in the larvae were homogenously distributed in the control organisms and no consistent spatial distribution could be observed in the exposed larvae (Figure 1). Therefore all further analyses are based on the mean total Cu body burden of the whole larva, excluding larva/air mixture pixels. Due to this homogeneous Cu distribution, the Cu concentration in the larva measured at a lower resolution (20 × 20 μm) could be used without bias. The mean absolute difference in Cu concentration between two larvae exposed to the same conditions was 1.5 μg/g DW larvae (SD: 0.86, n = 7). When data was available for two larvae of the same treatment, the average was taken for further analysis. All Cu body burdens ([Cubb]) are reported in the SI (see Table S2). The Cu body burden varied between 1.1 and 27.6 μg Cu/g DW larvae across all treatments with a mean control value over all DOC/salinity combinations of 2.5 μg Cu/g DW larvae (SD: 0.9, n = 9). In the larva measured at (sub)micrometer resolution level, Cu was spatially heterogeneously distributed at a resolution of 1 × 1 μm and 200 × 200 nm as can be seen in Figure 2. The Cu accumulation was higher along the edges of the larva and a clear Cu hotspot in the center of the hinge could be observed.

Figure 3. A: predicted [Cubb] (Model 3: log10 [Cubb] = −3.56 + 0.129 × [Cuext] − 4.66 × 10−3 × [Cuext]2 − 0.123 × [DOC] + 0.276 × salinity −4.43 × 10−3 × salinity2) compared to observed [Cubb] (n = 29), all but one value differ less than a factor 2 (dotted outer lines) between predicted and observed [Cubb]; B: the predicted effect of a change in DOC (0.8, 2, or 3.2 mg DOC/L, nominal middle and corner points of the experiment) and [Cuext] on [Cubb] at a fixed salinity of 30 psu according to model 3; C: the predicted effect of a changing salinity (25, 30, and 35 psu, nominal middle and corner points of the experiment) and [Cuext] on [Cubb] at a fixed [DOC] of 2 mg/L according to model 3.

Figure 2. High resolution SR-XRF image of Cu distribution in a mussel larva exposed to 10 μg Cu/L, darker regions indicate a higher Cu concentration (see also legend on the right); A: a whole larva at a pixel resolution of 1 × 1 μm; B: a detail of the same larva at a pixel resolution of 200 × 200 nm identical as the rectangle indicated in A.

Based on the statistical analysis it is concluded that the total external dissolved aqueous copper concentration ([Cuext]) alone can explain 46% (SI; Table S3) of the variation in the observed [Cubb] values. Backward selection from the full model including [Cuext], [DOC], salinity, interactions and quadratic effects resulted in model 2 (significant factors only, R2 adj =0.70, n = 29, AIC = −6.3) and model 3 (lowest AIC, R2 adj = 0.72, n = 29, AIC = −7.5). Both models (M.2 and M.3) can accurately predict the Cu body burden (Figure 3A) based on total external dissolved aqueous copper concentration. An increase in [Cuext] significantly increases [Cubb]. Addition of DOC significantly reduces Cu accumulation in mussel larvae

(Figure 1; Figure 3B) and salinity has a nonlinear effect on Cu accumulation with a maximum Cu accumulation at 32 psu (Figure 3C). The standard error, P-value and proportion explained variation of the individual terms is provided for each model in the SI (Table S3) log10[Cubb] = − 3.46 + 6.83 × 10−2[Cuext] − 0.127 × [DOC] + 0.275 × salinity − 4.39 × 10−3 × salinity 2 702

(M.2)

dx.doi.org/10.1021/es4024699 | Environ. Sci. Technol. 2014, 48, 698−705

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found by Rosen et al.9 but comparable to the concentrations found in our study. The reason behind this difference is unclear but may be due to differences in the drying protocol (air drying compared to freeze-drying). This difference in dry weight may have resulted in a much higher Cu concentration per unit of larval dry weight reported in their study.9 In the present study, mussel larvae were immediately dehydrated after the exposure and subsequently embedded in HMDS. This allowed, to our knowledge for the first time, measurements of Cu accumulation in individual mussel larvae. Additional experiments have confirmed that this sample preparation technique does not alter the Cu body burden in mussel gills when the Cu body burdens are compared to conventionally oven-dried gills (and ICP-MS, SI Figure S7). Models based on total dissolved copper indicate that both [DOC] and salinity have a significant effect on Cu accumulation (model 2 and 3). Reduced Cu accumulation with increasing DOC concentrations was expected, as DOC is known to reduce the accumulation and toxicity of Cu through the formation of Cu−DOC complexes.9,18 For example model 3 predicts a 43% decrease in Cu accumulation when [DOC] increased from 1 to 4 mg DOC/L at a dissolved Cu concentration of 8 μg/L. According to the BLM, an increase in salinity should increase the complexation of Cu with anions in the water, increase the ionic strength of the water and increase the competition with cations at the biotic ligand, therefore reducing the bioavailability of Cu. However, in the present study a nonlinear effect of salinity on Cu accumulation was observed with a steep increase in Cu accumulation up to 32 psu. Exposure to higher salinities reduced Cu accumulation in the larvae. This suggests that a change in larval physiology rather than a change in competition from cations caused the salinity-dependent alterations in Cu accumulation, certainly in the 23−32 psu range. According to Grosell et al.,13 physiology is important as changing salinity alters the osmolarity of the water and Cu toxicity is minimal at iso-osmotic conditions for osmoregulating organisms. However, mussel larvae are osmoconforming; therefore salinity should influence their physiology in a different way. The salinity where Cu accumulation is the highest corresponds closely to the salinity of 34.8 psu at which M. galloprovincialis larvae have been reported to develop optimally.45,46 This could indicate that a better development, and presumably higher metabolism, increases Cu uptake. Further research is needed to elucidate this relation. Although salinity explained only 7.1% of the variation in our experiment and the effect of salinity on Cu accumulation is less pronounced than the effect of DOC (21% of the variation explained, Figure 3, SI Table S3), salinity should not be neglected when studying Cu accumulation in mussel larvae. Cu2+ activity is, according to the BLM principles, the key determinant of Cu accumulation and adverse effects to aquatic organisms.2 Considering the whole mussel larva as a biotic ligand, our accumulation data can indeed be modeled more accurately using only Cu2+ activity instead of only total dissolved Cu (R2 adj. increased from 0.46 to 0.70). This indicates that, to some extent, the variation in accumulation is due to variations in Cu bioavailability. This is most probably the case for DOC as this effect is eliminated when modeling with Cu2+ activity instead of total dissolved Cu. Some studies suggest that Cu(CO3)22‑, Cu(OH)+ or Cu−DOC complexes can also be, partly, responsible for copper toxicity.1,11,47,48 In the present study, although Cu accumulation in mussel larvae is best predicted by Cu2+ activity, an influence of Cu(CO3)22− or

log10[Cubb] = − 3.56 + 0.129 × [Cuext] − 4.66 × 10−3 × [Cuext]2 − 0.123 × [DOC] + 0.276 × salinity − 4.43 × 10−3 × salinity 2

(M.3)

With [Cubb] as μg/g dry weight larvae, [Cuext] as μg/L, DOC as mg/L and salinity as psu. The logarithm of Cu2+ activity (log10{Cu2+}) by itself explains 70% of the variation in Cu body burden. All terms in the model (M.4) with the lowest AIC were significant (SI; Table S3, R2 adj. = 0.74, n = 29). An increase in {Cu2+} is associated with an increase of the Cu body burden (P < 0.001), the salinity (psu) has a similar effect on Cu accumulation as the models based on total dissolved Cu. 90% of the predicted Cu body burdens are within a factor 2 of the observed Cu concentrations (SI Figure S6). log10[Cubb] = − 0.163 + 0.237 × LOG10{Cu 2 +} + 0.251 × salinity − 4.04 × 10−3 × salinity 2

(M.4)

With [Cubb] as μg/g dry weight larvae, {Cu2+} as mol/L and salinity as psu.



DISCUSSION This is, to our knowledge, the first study to measure the concentration and spatial distribution of Cu in individual mussel larvae. The present study indicates that synchrotron radiation X-ray fluorescence (SR-XRF) spectroscopy is a useful tool to determine the concentration and distribution of Cu in small organisms with a submicrometer spatial resolution. The Cu distribution in the larvae revealed no consistent spatial variation at a resolution level of 10 × 10 μm (Figure 1). Therefore, the statistical analyses were performed on the mean Cu concentration in the whole larvae and the entire larva was considered as potential biotic ligand. However, at high spatial resolution level (1 μm or less, Figure 2), Cu in an exposed larva seemed to be heterogeneously distributed. At the moment it is unclear if this heterogeneity is consistent over several larvae. Because of the exponential increase in measuring time when the resolution is increased, it was at the moment not feasible to perform these high resolution measurements on all larvae in our experimental design. The submicrometer scale XRF measurements offer unprecedented possibilities for future research regarding the biological implications of spatially resolved metal accumulation in small biological samples like these mussel larvae and smaller certainly if technological advances reduce the measuring time. Cu accumulation increased with increasing total external dissolved aqueous Cu concentration. However, the Cu body burdens in the present study were a factor 2.9 lower than the concentrations found by Rosen et al.9 (calculated based on the mean ER50 (49.2 μg Cu/g DW) compared to calculations made by model 3 (17.1 μg Cu/g DW). The dry weight of the larva used in that study ranged approximately between 40 and 70 ng. This is a factor 1.7−4.5 lower compared to the larva dry weight values found in other studies of 134 ng,42 182 ng43 (at 100 μm shell length, which is approximately the length of a 48 h old D-larva) or between 120 and 160 ng.44 The Cu body burdens found by Geffard et al.44 are also lower than those 703

dx.doi.org/10.1021/es4024699 | Environ. Sci. Technol. 2014, 48, 698−705

Environmental Science & Technology



Cu(OH)+ on Cu accumulation cannot be excluded due to high covariation between the Cu species. Including salinity as predictor improves the model fit (significant and lower AIC, model 4). According to speciation calculations in Visual Minteq, the effect of salinity on the Cu2+ activity is small in the concentration range studied, with an increase in Cu2+ activity between 5 and 20% between the lowest and highest salinity depending on [DOC]. This increase in Cu2+ activity may be the result of an increased competition between Cu and cations (e.g., calcium) at DOC binding sites.49 According to model 4 a 20% increase in Cu2+ activity would result in an increased accumulation of maximum 4% and cannot account for the 46% increase in Cu accumulation that was observed between 25 and 32 psu (based on model 3, at [Cuext] = 8 μg/L). Therefore it is not surprising that salinity reduced the AIC of the regression model (4) and is considered important for predicting the copper accumulation based on Cu2+ activity. This model (4) indicates that salinity altered Cu accumulation in a similar way as model 2 or 3 (37% increase in Cu accumulation between 25 and 32 psu). Contradictory to our results, Cu accumulation should, according to the BLM, decrease with increasing salinities at the same Cu2+ activity and [DOC] due to increased competition at the biotic ligand. Thus, competitive effects cannot explain the observed changes in Cu accumulation in the present study. An altered physiology due to salinity changes is probably at the basis of the observed effect of salinity on Cu accumulation.



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ASSOCIATED CONTENT

S Supporting Information *

Details on experimental design, XRF measurements and Cu accumulation in mussel larvae. This material is available free of charge via the Internet at http://pubs.acs.org.



Article

AUTHOR INFORMATION

Corresponding Author

*Phone: +32 9264 3707; fax: +32 9264 3766; e-mail: David. [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS D.D, E.V., and J.G. are recipients of a Ph.D. grant provided by the Flemish Institute for the Promotion of Scientific and Technological Research in Industry (IWT, Belgium). Funding was obtained from the European Copper Institute and International Copper Association and from Ghent University “Concerted Research Action” (GOA) fund. Dr. Giovanni Libralato (Ca’Foscari University of Venice) is gratefully acknowledged for providing the mussels. Parts of this research were carried out at the synchrotron light sources DORIS-III (BL L) and PETRA-III P06) at DESY, a member of the Helmholtz Association (HGF). Funding was obtained from the European Copper Institute, International Copper Association and the BoF Ghent University Research Fund. 704

dx.doi.org/10.1021/es4024699 | Environ. Sci. Technol. 2014, 48, 698−705

Environmental Science & Technology

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